Proteins must fold correctly in order to attain biological function. Concurrently, protein aggregation and misfolding are key contributors to many devastating human diseases such as Alzheimer's disease, prion-mediated infections, type II diabetes, and cystic fibrosis. While "conventional" molecular chaperones assist protein folding by promoting the "forward" folding or preventing protein aggregation, they are unable to promote the disaggregation of already aggregated proteins such as amyloids, which are associated with certain human diseases. Bacterial CIpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, form large ring-like structures, and belong to the Hsp100 family of ATPases associated with diverse cellular activities (AAA+). Unlike any other chaperone, including other members of the Hsp100 family, CIpB/Hsp104 has the remarkable ability to promote the disaggregation of already aggregated, stress-damaged proteins. The underlying mechanism is currently unknown due to the lack of high-resolution structural information. The long-term objective is to understand the molecular mechanism by which members of the CIpB/Hsp104 family promote the disaggregation of stress-damaged proteins. The goals of this research will be pursued through the following specific aims: (1) to solve the high-resolution crystal structure of CIpB/Hsp104 using X-ray crystallography, (2) to elucidate the three-dimensional structure of the 0.6-MDa CIpB/Hsp104 assembly by cryo-electron microscopy, and (3) to determine the structural basis by which CIpB/Hsp104 recognizes and binds model substrates. These studies will be complemented by mutational and biochemical experiments to test the hypotheses inferred from these structures. The combination of these approaches will provide a detailed mechanistic understanding of the structure-function relationship of CIpB/Hsp104, and may inspire the design of novel technology that could lead to a potential cure for human prion and amyloid diseases.